Identification of germline transcriptional regulatory elements in Aedes aegypti

The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UD^MEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference

Targeting the X Chromosome during Spermatogenesis Induces Y Chromosome Transmission Ratio Distortion and Early Dominant Embryo Lethality in Anopheles gambiae

We have exploited the high selectivity of the homing endonuclease I-PpoI for the X-linked Anopheles gambiae 28S ribosomal genes to selectively target X chromosome carrying spermatozoa. Our data demonstrated that in heterozygous males, the expression of I-PpoI in the testes induced a strong bias toward Y chromosome–carrying spermatozoa. Notably, these male mosquitoes also induced complete early dominant embryo lethality in crosses with wild-type females. Morphological and molecular data indicated that all spermatozoa, irrespectively of the inheritance of the transgene, carried a substantial amount of I-PpoI protein that could attack the maternally inherited chromosome X of the embryo. Besides the obvious implications for implementing vector control measures, our data demonstrated the feasibility of generating synthetic sex distorters and revealed the intriguing possibility of manipulating maternally inherited genes using wild-type sperm cells carrying engineered endonucleases

A Toolbox of Aedes Aegypti Germline Regulatory Elements for the Development of Genetic Drive Systems and Population Control

Experiments in Drosophila have demonstrated that Medea-like genetic drive systems are engineer-able and can permit invasion of synthetic transgenes in wild populations. Similar accomplishments have also been made in Anopheles strains using engineered HEG-based gene drive systems. Synthetic circuits that generate a novel selfish phenotype typically require efficient germline expression of transgenes. To enable the transfer of this technology to Aedes mosquitoes, we have characterized a repertoire of promoters for expression of transgenes in germline tissues. Candidates were selected on the basis of expression data from a NGS transcriptome study and on evolutionary relationships to known maternal genes. The expression of eGFP driven by five Aedes aegypti and one known Anopheles gambiae germline specific regulatory elements was analyzed in transgenic reporter strains. Overall, the tools represent a significant expansion of regulatory elements for germline manipulation in this mosquito. I will describe the assorted expression profiles and discuss possible downstream applications

Sex separation strategies: past experience and new approaches

<p>Abstract</p> <p>The success of the sterile insect technique (SIT) and other genetic strategies designed to eliminate large populations of insects relies on the efficient inundative releases of competitive, sterile males into the natural habitat of the target species. As released sterile females do not contribute to the sterility in the field population, systems for the efficient mass production and separation of males from females are needed. For vector species like mosquitoes, in which only females bite and transmit diseases, the thorough removal of females before release while leaving males competent to mate is a stringent prerequisite. Biological, genetic and transgenic approaches have been developed that permit efficient male-female separation for some species considered for SIT. However, most sex separation methods have drawbacks and many of these methods are not directly transferable to mosquitoes. Unlike genetic and transgenic systems, biological methods that rely on sexually dimorphic characters, such as size or development rate, are subject to natural variation, requiring regular adjustment and re-calibration of the sorting systems used. The yield can be improved with the optimization of rearing, but the scale of mass production places practical limits on what is achievable, resulting in a poor rearing to output ratio. High throughput separation is best achieved with scalable genetic or transgenic approaches.</p

Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata

Homing-based gene drives are novel interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate two different mechanisms by which CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way

Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive.

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals

Core commitments for field trials of gene drive organisms

We must ensure that trials are scientifically, politically, and socially robust, publicly accountable, and widely transparent. Gene drive organisms (GDOs), whose genomes have been genetically engineered to spread a desired allele through a population, have the potential to transform the way societies address a wide range of daunting public health and environmental challenges. The development, testing, and release of GDOs, however, are complex and often controversial. A key challenge is to clarify the appropriate roles of developers and others actively engaged in work with GDOs in decision-making processes, and, in particular, how to establish partnerships with relevant authorities and other stakeholders. Several members of the gene drive community previously proposed safeguards for laboratory experiments with GDOs (1) that, in the absence of national or international guidelines, were considered essential for responsible laboratory work to proceed. Now, with GDO development advancing in laboratories (2–5), we envision similar safeguards for the potential next step: ecologically and/or genetically confined field trials to further assess the performance of GDOs. A GDO’s propensity to spread necessitates well-developed criteria for field trials to assess its potential impacts (6). We, as a multidisciplinary group of GDO developers, ecologists, conservation biologists, and experts in social science, ethics, and policy, outline commitments below that we deem critical for responsible conduct of a field trial and to ensure that these technologies, if they are introduced, serve the public interest. Includes Supplementary materials

Author Correction: Improved reference genome of the arboviral vector Aedes albopictus

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